CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells

DNA is routinely subject to exogenous or endogenous sources of damage (1). Therefore, cells have evolved complex surveillance mechanisms, known as cell-cycle checkpoints, to tightly regulate the cell cycle and maintain genomic integrity (2, 3). In normal cells, these checkpoints are activated in response to DNA damage, so that cell-cycle progression can be coordinated with the detection and repair of damaged DNA to prevent genomic instability. In cancer cells, these cell-cycle checkpoints are frequently deregulated, leading to high levels of replication stress and genomic instability, an enabling characteristic of cancer (4, 5).

Replication stress is often described as the slowing or stalling of replication fork progression during DNA synthesis and is caused by a number of different factors (6, 7). As a result of replication fork stalling, DNA polymerases become uncoupled from DNA helicases, which continue to unwind the DNA. This DNA unwinding generates stretches of ssDNA that are protected by binding of replication protein A (RPA; 8). RPA recruits a number of replication stress response proteins and is reliant on the ATR–CHK1 pathway to initially stabilize replication forks and delay the onset of mitosis until replication is resumed and completed (7, 9). Checkpoint kinase 1 (CHK1) is a serine/threonine kinase and a critical component of several cell-cycle checkpoints acting through multiple mechanisms, including replication fork stabilization and repair (10, 11). Cancer cells with defects in cell-cycle control, including those with high levels of replication stress, may therefore be more dependent on cell-cycle checkpoints and specifically CHK1 for survival (12). Such cancer-specific dependency could be exploited for therapeutic gain, making CHK1 an attractive anticancer target.

Preclinical validation of CHK1 as a cancer drug target, using siRNA and chemical tools, has led to the development of a number of CHK1 inhibitors (13), including the highly selective, potent, and orally bioavailable clinical drug candidate SRA737 (14, 15), which is undergoing evaluation in phase I clinical trials (www.clinicaltrials.gov; NCT02797977 and NCT02797964). CHK1 inhibitors have been shown to potentiate a number of chemotherapeutic agents (16), in particular gemcitabine (17, 18), and demonstrate single-agent activity in cancer subtypes with high levels of replication stress (17, 19, 20). Despite these observations, the underlying genetic determinants of sensitivity to clinically relevant CHK1 inhibitors remain unclear and there are currently no clinically validated biomarkers for patient selection. The identification of genes that are synthetically lethal in combination with CHK1 inhibition could lead to novel drug combination studies and potential predictive biomarkers of CHK1 inhibitor sensitivity. This may allow the identification of sensitive patient populations and aid the clinical application and evaluation of CHK1 inhibitors.

Synthetic lethality occurs when the simultaneous loss of function of two gene products results in cell death but the loss of either alone does not (21). Using the clinical drug candidate SRA737, we performed a large-scale synthetic lethal siRNA screen in human cancer cells to identify those gene products whose loss is synthetically lethal with CHK1 inhibition. Here, we show that siRNAs targeting POLA1, POLE, and POLE2, which encode subunits belonging to the B-family of DNA polymerases, are synthetically lethal in combination with CHK1 inhibition in multiple human non–small cell lung cancer (NSCLC) and colorectal cancer cell lines, suggesting a new potential treatment approach. Moreover, combinatorial depletion of the B-family DNA polymerases and small-molecule CHK1 inhibition resulted in increased replication stress, DNA damage, and cell death. Pharmacologic blockade of both B-family DNA polymerases and CHK1 led to synergistic inhibition of cancer cell proliferation. Furthermore, we show that low POLA1 and POLE protein expression may represent biomarkers for single-agent CHK1 inhibitor sensitivity.

Human cell lines were obtained from the ATCC, except SKLU1, which was obtained from the Health Protection Agency Culture Collections (United Kingdom). Lines were validated by DNA profiling and confirmed free of Mycoplasma species by PCR using the VenorGeM Mycoplasma PCR Detection Kit (Minerva Labs). All cell lines were purchased more than a year prior to the experiments and were propagated for less than 3 months after thawing. All experiments were performed within 10 passages after thawing. NSCLC cell lines were cultured in RPMI1640 supplemented with 10% FBS and colorectal cancer cell lines were cultured in DMEM with 5 mmol/L l-glutamine, 1% NEAA, and 10% FBS. HBEC3-KT cells were cultured in keratinocyte SFM serum–free medium supplemented with human recombinant EGF (5 ng/mL) and bovine pituitary extract (30 μg/mL). Cells were incubated at 37°C in a humidified atmosphere at 5% CO₂.

SRA737 was synthesized in-house at The Institute of Cancer Research (14, 15). Aphidicolin and CD437 were purchased from Sigma-Aldrich, MK-8776 from Selleck Chemicals, and gemcitabine from Eli Lilly. Stock solutions of SRA737, MK-8776, aphidicolin, and CD437 were prepared in DMSO and a stock solution of gemcitabine was prepared in 0.9 % NaCl solution. All stock solutions were stored at −80°C.

Total cell lysates were prepared as described previously (22) except that NP40 was increased to 0.3%. Protein concentration was determined using a bicinchoninic acid assay. Cell lysates (10–50 μg) were mixed in Laemmli sample buffer and heated for 3 minutes at 95°C. Proteins were separated by SDS-PAGE on precast 3% to 8% tris-acetate or 10% tris-glycine gels (Invitrogen), at 150 V for 60 to 90 minutes. Proteins were then transferred onto polyvinylidene difluoride membranes (Merck Millipore) at 100 V for 90 minutes. Membranes were incubated in 5% milk or 5% BSA in TNT buffer [50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, and 0.1% Tween 20] for 1 hour at room temperature and then overnight at 4°C with primary antibodies: POLA1 (ab31777), POLE (ab134941), POLE2 (ab57298), CHK1 pS345 (CST-2348), CHK1 (SC-8408), RPA32 (ab125681), cleaved PARP (CST-9541), and GAPDH (ab8245). Membranes were then washed and incubated with horseradish peroxidase–conjugated secondary antibodies (mouse/rabbit) for 1 hour. Proteins were visualized using enhanced chemiluminescence (Pierce Biotechnology) and hyperfilm (GE Healthcare).

The Dharmacon druggable genome siRNA library (Dharmacon, GE Healthcare Life Sciences) consists of siRNAs (four pooled siRNAs per gene), which target the expression of 7,593 genes that encode known drug targets or potentially druggable proteins. Of these 7,593 genes, 6,371 (84%) were screened, which included the kinase, phosphatase, and drug targets subsets. Dharmacon druggable human genome siRNA library master plates (384-well) containing lyophilized siRNA were resuspended in RNAse-free water to a final concentration of 5 μmol/L. Using the automated Echo 550 Liquid Handler (Labcyte), 0.5 μL of siRNA was then transferred from a master plate to six 96-well daughter plates, to give a final assay concentration of 25 nmol/L. siRNAs were introduced into cells by reverse transfection; 50 μL of HiPerFect in Opti-MEM (Invitrogen) was added to each well at a final assay concentration of 0.3% and incubated for 20 minutes. Cells (50 μL), at optimum seeding density, were then added to each well, incubated for 48 hours at 37°C and treated with SRA737 at the maximum sublethal dose (0.4 μmol/L or 0.8 μmol/L for A549 and SW620, respectively) or DMSO vehicle (0.004% or 0.008% for A549 and SW620, respectively) in 100 μL medium followed by incubation for 84 hours at 37°C. Plates were fixed with 10% trichloroacetic acid and stained with 0.4% sulphorhodamine-B (SRB) in 1% acetic acid. SRB was solubilized with 10 mmol/L Tris base and plates were read at 490 nm using a Wallac Victor 1420 multilabel counter (Perkin-Elmer). Three independent biological repeats of the screen were carried out for each cancer cell line.

A Z'-factor was calculated for the entire screen to ensure a wide assay window for hit identification, which was maintained (23). Robust Z-scores were calculated to identify hits from the screen. The difference in robust Z-score, with and without SRA737, was calculated for each siRNA in each biological repeat and averaged. Hits were defined as those genes with a difference in robust Z-score greater than or equal to three, with the most significant differences given higher priority. Additional selection criteria for further hit evaluation were (i) a minimal effect of siRNA alone on cell number and (ii) commonality to both cell lines.

Hits were validated using pooled siRNA (Dharmacon) and individual siRNAs (Qiagen). HiPerFect was used at 0.3% (A549, SW620 cells) or 0.2% (Calu-6, NCI-H1975, RKO, and HCT116 cells). HiPerFect in Opti-MEM (40 μL) was added to 10 μL siRNA in RNAse-free water in each well and incubated for 20 minutes. Cells (50 μL) were then added at the optimal seeding density and incubated for 48 hours at 37°C. Plates were treated with SRA737 (0.4 μmol/L or 0.8 μmol/L for A549 and SW620, respectively) or DMSO vehicle (0.004% or 0.008% for A549 and SW620, respectively) in 100 μL medium and incubated for 84 hours at 37°C. Plates were fixed and stained with SRB and read and analyzed as described above.

Knockdown experiments were carried out in 6-well plates using reverse transfection. siRNA and HiPerFect were added at 3 × the final assay concentrations (500 μL/well) and incubated for 20 minutes. Next, 500 μL of cells were added to each well and incubated for 6 hours at 37°C. Medium (2 mL) was then added to each well to make 1 × siRNA and HiPerFect concentration and cells were incubated for 48 hours at 37°C. Protein expression levels were determined by Western blot analysis.

Cells were transfected with nonlethal concentrations of siRNA as described above. Plates were treated with 0.01–1 μmol/L SRA737 or 0.1–10 μmol/L MK-8776 for 84 hours at 37°C. Plates were fixed and stained with SRB and read and analyzed as described above. Data were analyzed using GraphPad Prism 6.0.

Cells were plated in 96-well plates at optimum seeding densities and incubated for 36 hours at 37°C. Plates were then treated with single-agent SRA737 or aphidicolin, or a combination of the two, at a 1:1 ratio of each GI₅₀ (previously determined) and incubated for four doubling times at 37°C. Plates were fixed and stained with SRB and read and analyzed as described above. Data were analyzed using the Chou and Talalay method to generate a combination index (24).

Cells were plated in 96-well clear-bottom opaque-sided plates, incubated for 36 hours at 37°C, and subsequently treated with a GI₅₀ concentration of aphidicolin or SRA737 or a combination of the two, followed by incubation for 24 hours at 37°C. Gemcitabine at a final concentration of 200 nmol/L was added as control. Cells were then fixed in 4% formaldehyde for 20 minutes and permeabilized with 0.25% Triton X-100 for 10 minutes. Next, the fixed cells were blocked with 3% FBS in TNT for 1 hour, incubated overnight at 4°C with a γH2AX antibody (Upstate #05636) at 1:500 and subsequently washed with PBS. The secondary antibody (Alexa Fluor 488 goat anti-mouse IgG, A11001 Invitrogen) was added at 1:1,000 for 1 hour, cells were then washed, DAPI was added at 1:1,000 for 5 minutes, and cells were washed again before adding 200 μL PBS per well. Images were acquired using an IN Cell Analyser 2200 and were analyzed using an IN Cell Analyser 1000 Workstation (version 3.7.2, GE Healthcare Life Sciences).

A549 cells were plated onto 22 × 22 mm coverslips (0.16–0.19 mm thickness) in 6-well plates at 5 × 10⁵ cells per well and incubated for 24 hours. Medium was removed and cells were preincubated with 1.9 mL fresh medium for 1 hour, after which, 100 μL of each drug was added at the required concentration. Cells were incubated for a further 24 hours. Medium was then removed, cells washed twice with PBS, and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. PFA was removed and cells washed three times with PBS followed by permeabilization in TBS containing 3% Triton-X100 for 15 minutes at room temperature. Permeabilized cells were washed twice in TBS before blocking in TBS containing 0.1% Tween and 2% BSA (TBST/BSA) for 1 hour at room temperature. Cells were incubated with Anti-phospho-Histone H2A.X (Ser139) mouse primary antibody (EMD Millipore) at 1:2,000 in TBST/BSA overnight at 4°C. Cells were then washed three times in TBST/BSA before incubation in Alexa Fluor 488 donkey anti-mouse secondary antibody (Invitrogen) at 1:1,000 for 1 hour in the dark at room temperature. Cells were washed twice with TBST/BSA and then once with TBS. During this final TBS wash, two drops of NucBlue Live ReadyProbes Reagent (Invitrogen) were added per 1 mL of TBS for each coverslip and incubated at room temperature in the dark for 20 minutes. After nuclear staining, slides were mounted using ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and left to dry overnight in the dark before imaging.

Fixed sample slides were fitted onto an ASI Motorised Stage (ASI) and visualized using an Olympus IX71 microscope with PlanApo 100x OTIRFM-SP 1.45 NA lens mounted on a PIFOC z-axis focus drive (Physik Instrumente), with illumination using LED light sources (Cairn Research Ltd) with appropriate filters (Chroma). Samples were visualized using a QuantEM (Photometrics) EMCCD camera, and the entire system was controlled with Metamorph Software (Molecular Devices). 3D-maximum projections of volume data were calculated from 31 z-plane images, each 0.2 μm apart. Data were subsequently analyzed using Autoquant Software (MediaCybernetics). All 3D image stacks were subjected to blind 3D deconvolution before analysis. Number and intensity of foci were calculated from all foci present within ≥30 cells for each sample examined.

To identify gene products whose loss is synthetically lethal with CHK1 inhibition, we performed an unbiased, large-scale siRNA screen with the developmental clinical drug SRA737 and the Dharmacon druggable human genome siRNA library in human A549 NSCLC and SW620 colorectal cancer cells. These cell lines were selected because of their relatively low sensitivity to SRA737 (Supplementary Fig. S1), similar growth characteristics (i.e., doubling time and plating efficiency), and ease of transfection. In total, 6,371 genes were screened with or without SRA737 in both cells lines, with three independent biological repeats per cell line.

The screen was robust and reproducible, as shown by the fact that the WEE1 siRNA–positive control (25) consistently sensitized A549 and SW620 cells to SRA737 and calculated Z’ factors were generally >0.5 (Fig. 1A and B; refs. 23, 26). Any plates with Z'- factors < 0.5 were rejected from the analysis and rerun. Hits were identified as those genes with a difference in robust Z-score under control (0.004%–0.008% DMSO vehicle) and test (SRA737) conditions of ≥3 (Fig. 1C and D; Supplementary Tables S1 and S2).

A number of the hits with the highest difference in robust Z-score encoded products that were associated with DNA replication (Fig. 1C and D), including MCM6 (mini-chromosome maintenance complex component 6), RPA1 (replication protein A1), POLA1 (DNA polymerase α, catalytic subunit), POLE (DNA polymerase ϵ, catalytic subunit), and POLE2 (DNA polymerase ϵ, accessory subunit). POLA1, POLE, and POLE2 encode subunits of B-family DNA polymerases, responsible for high-fidelity DNA replication (27), and were identified as hits in both the A549 and SW620 cancer cell lines. POLA1, POLE, and POLE2 met all the hit prioritization criteria (see Materials and Methods section) and were therefore selected for further validation.

To confirm that knockdown of POLA1, POLE, and POLE2 is synthetically lethal in the context of pharmacologic CHK1 inhibition, cells were transfected with multiple siRNAs, each targeting a different region of the mRNA of each hit gene. Allstars negative (nontargeting) and positive death control siRNAs (Qiagen) were used as controls and to monitor toxicity of the transfection reagents and the transfection efficiency, respectively. Both A549 and SW620 human cancer cells were significantly sensitized to SRA737 by siRNA-mediated depletion of POLA1 and POLE2 (Fig. 2A and B), while only A549 cells were also significantly sensitized to SRA737 by transfection with POLE siRNA (Fig. 2A). Depletion of the cognate proteins by the siRNAs was confirmed even at the lowest concentrations tested (Fig. 2C and D).

To quantify the extent to which depletion of POLA1, POLE, or POLE2 could sensitize cancer cells to SRA737, we determined the GI₅₀ values for SRA737 in both A549 and SW620 cancer cell lines transfected with low concentrations of POLA1, POLE, and POLE2 siRNA. Knockdown of POLA1, POLE, or POLE2 significantly decreased the GI₅₀ for SRA737 in both cancer cell lines, resulting in between 5- to 16-fold sensitization to this CHK1 inhibitor drug (Fig. 3A–E). Importantly, we obtained similar results using MK-8776 (SCH 900776; ref. 28), a second CHK1 inhibitor from a different chemotype to SRA737 (15), thus increasing the likelihood that the effects seen represent a genuine and specific synthetic lethal interaction between pharmacologic inhibition of CHK1 function and depletion of B-family DNA polymerases (Supplementary Fig. S2A–S2C).

To confirm that the synthetic lethal interaction that we observed involved an on-target effect of SRA737, we performed CHK1 siRNA-knockdown studies in combination with aphidicolin, a tetracyclic diterpene antibiotic, which is a potent inhibitor of B-family DNA polymerases α, δ, and ϵ (29–31). We found that A549 cancer cells were indeed sensitized to aphidicolin treatment following siRNA-mediated knockdown of CHK1 (Supplementary Fig. S3A), with reduction in CHK1 protein expression confirmed by Western blot analysis (Supplementary Fig. S3B).

Next, we investigated if the synthetic lethal interaction between POLA1, POLE, or POLE2 knockdown and CHK1 inhibition could be seen in additional human NSCLC (NCI-H1975 and Calu-6) and colorectal cancer (HCT116 and RKO) cell lines. Encouragingly, all four additional cancer cell lines were significantly sensitized to SRA737 by POLE and POLE2 depletion (Supplementary Figs. S4A–S4D, S5A and S5B, and S6A and S6B). Furthermore, NCI-H1975 and Calu-6 cells were significantly sensitized to SRA737 by transfection with POLA1 siRNA (Supplementary Fig. S4A and S4B). Note, however, that in both HCT116 and RKO colorectal cancer cell lines, transfection with POLA1 siRNA alone markedly reduced cell number (Supplementary Fig. S4C and S4D), suggesting that these cell lines may have an absolute requirement for POLA1 and/or that knockdown was more efficient in RKO and HCT116 cells versus NCI-H1975 and Calu-6 NSCLC cells (Supplementary Figs. S5 and S6).

To confirm that the synthetic lethal interaction we observed was due to loss of both B-family polymerase and CHK1 catalytic activity, we adopted an orthogonal validation approach by performing combination studies using small-molecule inhibitors of both CHK1 and B-family DNA polymerases. We tested SRA737 in combination with aphidicolin in a panel of human cancer cell lines. We found that SRA737 and aphidicolin cotreatment was synergistic in eight of nine cancer cell lines tested (Fig. 4A; Supplementary Table S3), including A549 lung and SW620 colorectal cancer cells (Fig. 4B and C).

Clinical development of aphidicolin glycinate, a water-soluble analogue of aphidicolin, as an anticancer drug has been hampered by poor pharmacokinetics and toxicity (32). More recently, the structurally distinct retinoid-like agent CD437 has emerged as a DNA polymerase inhibitor with selectivity for α over other B-family polymerases (33). Reassuringly, as with aphidicolin, synergy was also observed between SRA737 and CD437 in both the A549 NSCLC and SW620 colorectal cancer cell lines (Fig. 4D and E). Importantly, the synergy observed between SRA737 plus aphidicolin or SRA737 plus CD437 was comparable with that seen with the developmental clinical combination of SRA737 plus the chemotherapeutic agent gemcitabine (Supplementary Fig. S7A–S7D and Supplementary Table S3).

In contrast to the results obtained in cancer cell lines, we found no synergy but only an additive effect for SRA737 combined with aphidicolin in the HBEC3-KT normal human bronchial epithelial cell line that is immortalized with CDK4 and hTERT (Supplementary Fig. S8; ref. 34). This would suggest that transformed cells may be more susceptible to the drug combination versus their nontransformed counterparts. Overall, our data demonstrate that pharmacologic inhibitors of CHK1 and B-family DNA polymerases combine synergistically to inhibit cancer cell proliferation.

We hypothesized that combined loss of CHK1 and B-family DNA polymerase function would lead to enhanced replication stress, DNA damage, and apoptosis. To test this, we examined RPA32 (Replication protein A 32 kDa subunit), which is phosphorylated at multiple sites in response to replication stress, an effect that can be visualized as a band shift on an immunoblot (7, 35–37). We observed an RPA32 band shift after siRNA-mediated depletion of POLA1 alone, indicative of replication stress, but this was not visible with depletion of POLE or POLE2 alone in the A549 or SW620 cell lines (Fig. 5A). However, all combined treatments of SRA737 plus POLA1, POLE, or POLE2 siRNA induced a band shift in RPA32 (Fig. 5A). Furthermore, pharmacologic inhibition of B-family polymerases with either aphidicolin or CD437 induced a detectable RPA band shift, which was further enhanced when combined with the CHK1 inhibitor SRA737 (Supplementary Fig. S9).

Persistent and unresolved replication stress can lead to increased DNA damage and cell death (38). We found significantly higher levels of the DNA damage marker γH2AX in five of six cancer cell lines cotreated with the CHK1 inhibitor SRA737 and the B-family DNA polymerase inhibitor aphidicolin compared with either treatment alone, as detected by immunofluorescence using an IN Cell Analyser (Fig. 5B). We confirmed that the increase in γH2AX signal was located in foci, which is indicative of direct DNA damage (Supplementary Fig. S10A–S10F). In addition, all combined treatments of SRA737 plus POLA1, POLE, or POLE2 siRNA (apart from SRA737 plus POLE2 depletion in SW620 cells) also caused an increase in the level of cleaved PARP (C-PARP), a marker of apoptosis, when compared with treatment with siRNA or SRA737 alone (Fig. 5A). Taken together, these data indicate that the combined inhibition of the DNA polymerases and CHK1 leads to enhanced replication stress, DNA damage, and apoptotic cell death.

On the basis of our results with depletion of B-family DNA polymerases, we hypothesized that cancer cells with lower levels of POLA1, POLE, and POLE2 protein expression would be more sensitive to CHK1 inhibition compared with those with higher levels of these polymerase subunits. To test this, we measured the level of basal protein expression of POLA1, POLE, and POLE2 in the panel of NSCLC and colorectal cancer cell lines (Fig. 6A; genetic status data Supplementary Table 4) and compared these data with GI₅₀ values for single-agent SRA737 treatment (Supplementary Table 5). We found a significant correlation between POLA1 and POLE protein expression and SRA737 sensitivity (P ≤ 0.01), and between POLE2 protein expression and SRA737 sensitivity (p ≤ 0.05; Fig. 6B), suggesting that low-basal expression of each of these proteins is indeed indicative of CHK1 inhibitor sensitivity. We also observed that the basal protein expression level of each of these polymerase subunits correlated with the levels of the others (Supplementary Fig. S11A–S11D), consistent with reports that their expression is coordinated (39, 40). In contrast, POLA1, POLE, and POLE2 basal protein expression did not correlate with sensitivity to the combination of SRA737 and aphidicolin (Supplementary Fig. S12A–S12C).

CHK1 inhibitors are currently in early clinical trials, both in combination with genotoxic agents and also as single agents (13, 16). However, insights into tumor cell vulnerabilities that increase sensitivity to CHK1 inhibitors and biomarkers of this phenotype have been lacking. We used an unbiased large-scale siRNA screen to identify genes that are synthetically lethal when depleted in combination with CHK1 inhibition. We found that the knockdown of subunits of the B-family of DNA polymerases, POLA1, POLE, and POLE2, sensitized human NSCLC and colorectal cancer cells to CHK1 inhibition. This synthetic lethal relationship was identified with the CHK1 inhibitor SRA737 and confirmed with the structurally distinct CHK1 inhibitor MK-8776. Moreover, in addition to validation with multiple siRNAs, the interaction was seen with two chemically distinct B-family DNA polymerase inhibitors, namely aphidicolin and CD437, thus demonstrating the importance of catalytic inhibition. Note that such agreement between two distinct chemotype inhibitors is consistent with best practice for chemical probe use and provides greater confidence, alongside the orthogonal application of genetic depletion as also seen here, that the synthetic lethal interaction involves on-target effects on catalytic function (41). Importantly, we have demonstrated that the synthetic lethal interaction between CHK1 and the B-family DNA polymerases leads to a synergistic pharmacologic effect and, moreover, we showed that this occurred in multiple human NSCLC and colorectal cancer cell lines. In addition, the synergy observed is comparable with that seen with the combination of SRA737 plus the chemotherapeutic agent gemcitabine that is currently undergoing clinical evaluation. Observing such robustness in the synthetic lethal interaction is very important and indicates that this effect is real and independent of the range of oncogenic abnormalities in the different cancer cell lines studied. The comparable synergy seen with inhibition of CHK1 plus B-family DNA polymerases and inhibition of CHK1 plus gemcitabine is encouraging, given that gemcitabine is typically considered as the most synergistic genotoxic drug for use in combination with CHK1 inhibition (18).

A recent large-scale analysis has shown that robustness in synthetic lethal interactions is enriched in protein–protein interaction pairs (42). B-family DNA polymerases are responsible for the high-fidelity replication of DNA (27). Several studies have reported a functional relationship between these DNA polymerases and CHK1. Thus, CHK1 has been shown to coimmunoprecipitate with DNA polymerase α, indicating a direct interaction between the two proteins (43). Some studies have indicated that DNA polymerase α is required for CHK1 activation (8, 44); conversely, other studies report that depletion of DNA polymerase α by siRNA leads to CHK1 activation (43). Our own data herein show that depletion of the B-family DNA polymerases leads to a robust increased dependence of cancer cells on CHK1 function.

We hypothesized that combined loss of CHK1 and B-family DNA polymerase function leads to enhanced replication stress, DNA damage, and apoptosis. This was confirmed by the presence of phosphorylated RPA, γH2AX foci, and PARP cleavage, respectively, which were all increased in human cancer cells following partial depletion of B-family DNA polymerase subunits combination with SRA737 treatment. Moreover, as mentioned, the synergy between SRA737 and aphidicolin or CD437 was comparable with that seen with the combination of SRA737 plus gemcitabine in the NSCLC cell lines tested. Aphidicolin and CD347 both bind to the nucleotide binding site of the DNA polymerase, thus directly inhibiting polymerase activity (31, 33). In contrast, gemcitabine inhibits the B-family DNA polymerases indirectly via two mechanisms: (i) depleting the pool of dNTPs available for DNA replication; and (ii) misincorporation into replicating DNA, which generates lesions capable of stalling the B-family DNA polymerases (45). The shared inhibition of the B-family of DNA polymerases, albeit through a different molecular mechanism, almost certainly underlies the synergy seen with all three agents when combined with CHK1 inhibition.

It would be interesting to determine if CHK1 inhibitors are also effective in combination with other agents capable of inducing replication stress. Recently, the neddylation inhibitor MLN4924, which stabilizes the rereplication factor CDT1 and potentially increases replication stress, has been shown to enhance the antitumor activity of the CHK1 inhibitor MK-8776, thus further supporting the hypothesis that inducing replication stress sensitizes cancer cells to CHK1 inhibitors (46). On the basis of these findings and our own data, we propose a possible model for how the combined pharmacologic inhibition of B-family DNA polymerases and CHK1 may increase replication stress, DNA damage, and apoptotic cell death (Fig. 7). This suggests that CHK1 is required to alleviate the replication stress induced by loss of the B-family DNA polymerase function, possibly by stabilizing stalled replication forks. Thus, loss of CHK1 when combined with depletion of B-family polymerases may cause intolerable levels of replication stress, DNA damage, and eventual cell death. Further work is required to establish if this is the case.

Germline and somatic mutations in DNA polymerase δ (POLD1) and ϵ (POLE) have been identified in a number of different cancers, particularly colorectal and endometrial (47). There are over 200 reported mutations in POLE occurring along the entire gene, of which, few have been fully characterized and, to date, none have been associated with loss of the protein, perhaps unsurprisingly as DNA polymerase function is essential for viability. Interestingly, the high-mutation rates or hypermutator phenotype associated with colorectal and endometrial cancer have been attributed in part to mutations in the exonuclease domains of the DNA polymerases, leading to loss of their proofreading capabilities (47). We show here low POLA1, POLE, and POLE2 protein expression correlate with sensitivity to CHK1 inhibition. This is possibly because cancer cells expressing the B-family DNA polymerases at low levels are more sensitive to increases in replication stress. Thus, low expression of these polymerases at the protein level could potentially be used as biomarkers to predict patient sensitivity to CHK1 inhibitor monotherapy, although this needs to be confirmed with a large panel of cell lines and using clinical samples. In contrast, POLA1 POLE, and POLE2 protein expression did not correlate with sensitivity to the combination of SRA737 and aphidicolin, perhaps because the pharmacologic inhibition of B-family DNA polymerases α, δ, and ϵ polymerase with aphidicolin overrides low-level expression of B-family polymerases as a biomarker for this phenotype.

It is interesting to compare our results with those obtained previously in relevant synthetic lethal screens. Hocke and colleagues (48) looked for genes that were synthetic lethal with ATR deficiency by screening an siRNA library corresponding to 288 DNA repair genes in a well-characterized genetic ATR-knockin model of DLD1 human colorectal cancer cells as compared with wild-type counterparts. They identified as the strongest hit POLD1, which encodes the DNA polymerase δ catalytic subunit. Reasoning that CHK1 is the major downstream effector of ATR, they used the staurosporine analog UCN-01 as an inhibitor of CHK1 and showed greater sensitivity in POLD1-depleted versus control cells. However, UCN-01 is known to be a nonselective inhibitor of CHK1 (49) and follow-up studies in additional colorectal cancer cell lines showed that POLD1-knockdown resulted in a much smaller degree of sensitization to UCN-01, suggesting that the original finding in DLD1 cells was probably a cell line–specific observation. Moreover, although siRNAs targeting POLD1 were included in our own screen, as well as the screen reported by Davies and colleagues (25) of siRNAs to cell cycle and DNA repair genes looking for synthetic lethality with the CHK1 inhibitor AR458323 (and which identified WEE1 as synthetic lethal), POLD1 was not identified as a hit in either of these screens. The conflicting results with POLD1 likely reflect differences in the cancer cell lines and/or the CHK1 inhibitors used in the studies. In addition, POLA1 and POLE, both hits in our own screen, were not identified as synthetically lethal hits in the screen by Hocke and colleagues (48) because siRNA against these genes proved lethal to DLD1 cells when applied on their own at 10 nmol/L. The findings we have reported here indicate that low concentrations of siRNA (less than 0.3 nmol/L) can reduce polymerase levels sufficiently to see synthetic lethality with CHK1, but still leave enough residual activity for cells to survive under control conditions.

In addition to our demonstration that low POLA1, POLE, and POLE2 protein expression predispose cells to sensitivity to CHK1 inhibition, previous studies have indicated that amplification and/or elevated expression of MYC family genes may lead to CHK1 inhibitor sensitivity in a number of different cancer types (50, 51). For example, c-MYC overexpression predicts response to single-agent treatment with the dual CHK1/CHK2 inhibitor LY2606368 in a panel of small-cell lung cancer cell lines. This is thought to be due to the increase in replication stress associated with MYC overexpression (51). In addition, SRA737 has been shown to be effective as a single agent in vivo in mouse models of Eμ-MYC–driven B-cell lymphoma and pediatric MYCN-driven neuroblastoma (14, 15). Therefore it is conceivable that overexpression of other oncogenes associated with increased replication stress, such as cyclin E or RAS (52), or replication-associated factors, which allow cancer cells to cope with replication stress, such as CDT1 (53), could lead to CHK1 inhibitor sensitivity. Interestingly, a recent study has demonstrated that cyclin F loss predisposes cells to CHK1 inhibition by inducing DNA replication catastrophe (54). Moreover, other direct markers of replication stress, such as pRPA, pCHK1, and ssDNA, may also predict for CHK1 inhibitor sensitivity.

In conclusion, we have shown that both siRNA depletion and pharmacologic inhibition of the B-family of DNA polymerases are synthetically lethal in combination with CHK1 inhibition in human lung and colorectal cancer cells. Combination studies revealed that the synergistic interactions between either of two B-family polymerase inhibitors aphidicolin and CD437 with the CHK1 inhibitor SRA737 were comparable with those seen with the promising combination of gemcitabine and SRA737 that is currently undergoing clinical evaluation. Thus, the combination of B-family DNA polymerase inhibition with SRA737 could form the basis, subject to further follow up research, of a potential new future therapeutic approach. The clinical development of the water-soluble aphidicolin analog, aphidicolin glycinate, was limited by its rapid clearance, low bioavailability, and severe toxicity at the injection site and is no longer being pursued (32). The more recent retinoid-like agent CD437 has not reached the clinic and also inhibits the retinoic acid receptor γ and potentially other off-targets as well as having nonoptimized pharmaceutical properties (33). Thus the evaluation of the therapeutic potential of a CHK1 inhibitor in combination with a B-family DNA polymerase inhibitor in animal models and patients must await the emergence of suitable B-family DNA polymerase drug candidates, which the current work encourages. Finally, we found that low POLA, POLE, and POLE2 protein expression could potentially be used as biomarkers to predict patient sensitivity to CHK1 inhibitor monotherapy, which could, subject to confirmation and further validation, facilitate the effective clinical use of CHK1 inhibitors as single agents.

R.F. Rogers, M.I. Walton, I. Collins, P.A. Clarke, M.D. Garrett, and P. Workman are current or past employees of The Institute of Cancer Research, which has a commercial interest in the discovery and development of CHK1 inhibitors, including SRA737, and operates a rewards to inventors scheme. They are also named as inventors on relevant patents. I. Collins is consultant for AdoRx Ltd, Epidarex LLP, and Enterprise Therapeutics Ltd, reports receiving a commercial research grant from 6th Element Capital, has ownership interest (including patents) in Monte Rosa Therapeutics AG, and has an unpaid consultant/advisory board relationship with Sierra Oncology. M.D. Garrett is a scientific advisory board member at Sierra Oncology, consultant at Centauri Therapeutics and Azeria Therapeutics, is a scientific advisory board member at NCL Technology Ventures, consultant at Kinsensus Limited, and has an unpaid consultant/advisory board relationship with CRUK Therapeutic Discovery Laboratories. P. Workman is an independent Director (Board) at Storm Therapeutics, is a consultant/advisory board member at Astex Pharmaceuticals, CV6 Therapeutics, and Nextechinvest, reports receiving a commercial research grant from Sixth Element Capital, Astex Pharmaceuticals, and Merck, has ownership interest in Storm Therapeutics, Chroma Therapeutics, and Nextechinvest, and has an unpaid consultant/advisory board relationship with the Royal Marsden NHS Foundation Trust and the Chemical Probes Portal. No potential conflicts of interest were disclosed by the other authors.

Conception and design: R.F. Rogers, M.I. Walton, I. Collins, P.A. Clarke, M.D. Garrett, P. Workman

Development of methodology: R.F. Rogers, P.A. Clarke

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.F. Rogers, D.L. Cherry, M.D. Garrett, P. Workman

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.F. Rogers, M.I. Walton, D.L. Cherry, M.D. Garrett, P. Workman

Writing, review, and/or revision of the manuscript: R.F. Rogers, M.I. Walton, I. Collins, P.A. Clarke, M.D. Garrett, P. Workman

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.F. Rogers

Study supervision: M.I. Walton, P.A. Clarke, M.D. Garrett, P. Workman

The authors thank Nicky Evans (Institute of Cancer Research, ICR) for editorial assistance, Dan Mulvihill and Karen Baker (School of Biosciences, University of Kent, Canterbury, United Kingdom) for help with confocal microscopy work and both Albert Hallsworth and Vladimir Kirchin (ICR) for their input on this project. P. Workman, M.D. Garrett, M.I. Walton, and I. Collins acknowledge program grant support from Cancer Research UK (CRUK grant numbers C309/A11566 and C2739/A22897) and support from the ICR (London, United Kingdom). P. Workman acknowledges additional grant support from the Wellcome Trust (212969/Z/18/Z) and Cancer Research UK (C35696/A23187) and he is a Cancer Research UK Life Fellow. R.F. Rogers acknowledges support from the Wellcome Trust (102359/Z/13/Z). M.D. Garrett and D.L. Cherry also acknowledge financial support from the University of Kent (Canterbury, United Kingdom, cc210/GM02), Kent Cancer Trust (SCP343), and the EB Hutchinson Charitable Trust (A2696). ICR authors acknowledge support from the CRUK Centre at the Institute of Cancer Research.

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